Summary Toxoplasma gondii is an obligate intracellular apicomplexan parasite causing severe opportunistic infections. Current drugs are prone to induce hypersensitivity, especially upon long-term use. In order to identify new drug targets, the P.I.?s lab focuses on deciphering cell biological processes wherein the parasite differs from the host. In this proposal the distinct structure addressed is the peripheral or apical annuli. The apical annuli comprise a cluster of 5-6 enigmatic structures containing Centrin2, which sit at the peri-apical end of the membrane skeleton known as the inner membrane complex (IMC). Annuli function is unknown and has not been studied. To pursue their function, the PI?s lab started to dissect the composition and architecture of the apical annuli by reciprocal proximity based biotinylation (BioID). Together with a recently reported protein unique to the annuli, Peripheral Annuli Proteins 1 (PAP1, which was also detected), the four new unique annuli proteins were named PAP2-5. In addition, BioID detected known IMC suture proteins. Validation of the annuli- suture connection by super resolution microscopy (SIM) showed that the annuli reside at the suture intersections between the cap- and central-alveoli. Moreover, SIM revealed that the annuli proteins are organized in concentric rings of 220-440 nm diameters, suggestive of a pore. The size and suture embedding are morphologically very similar to the appearance of ?ejectosomes? in the glaucophyte and crytophyte algae. Interestingly, these algae share with the Apicomplexa a newly recognized class of epiplastin proteins known as IMC proteins in Toxoplasma that form ?epiplastid? cytoskeletons. We hypothesize that the annuli are epiplastid spanning pores. Since the PAP repertoire is only conserved in the genomes of Apicomplexa dividing by internal budding, we surmise that apical annuli serve as conduits for nutrients and waste products across the mother?s IMC as this is maintained during internal budding and thus an obvious obstacle for free exchange. To directly test function, a direct KO of PAP4 was generated, which resulted in severe fitness reduction and loss of acute virulence in mice, demonstrating that the apical annuli are critical. The goal of this proposal is to map the key apical annuli function by pursuing the question at three levels: A. Genetically, by generating (conditional) PAP ablated parasites and phenotype dissection; B. Morphologically, by ultrastructure using IEM, CLEM and freeze-fracture EM (QFDEEM); C. Evolutionary, by assessing the annuli in Sarcocystis neurona which by endopolygeny and produces 64 daughters per division round, predicting an expanded annuli reportoire. Although our preliminary data align strongly with a pore function, alternative, possibly overlapping, functions include structural, vesicular trafficking, and signaling roles. The chosen assays can differentiate these functions. Upon completion we will have mapped the key function(s) and the architecture of the apical annuli that will illuminate a very understudied area of apicomplexan biology likely relevant to all epiplastid skeletons.